1. Field of the Invention
The present invention relates to a method for genetic manipulation in cell lines and organisms. More particularly, the invention relates to a method and components thereof for genetic manipulation without applying drug selections.
2. Description of the Prior Art
Since the recent completion of the human and mouse genome projects, genomic medicine becomes one of the fastest growing areas of biomedical research today. Enormous efforts are devoted to developing tools and technologies to decipher the human genetic code. However, how these three billion nucleotides of human genome carry out their multitudes of function is still largely remain unknown. The information of gene function is a prerequisite to delineate the therapeutic targets for disease diagnosis. Therefore, revealing the function of each gene and its role in the biological pathways will shed lights on the molecular mechanism of pathogenesis and in turn, leads to the development of effective therapeutic strategies. Due to the complexity of biological processes that form the basis of most diseases, the method of genome-wide genetic manipulation in model organisms is gaining a momentum in modern biological science.
Genetic alternation in established cell lines or the mouse genome has been the most frequently used strategy to dissect the structure and functions of genetic networks in mammals. Human and mouse gene share 99% of homology and many defects observed in knockout mice phenocopy those seen in human diseases. Hence, analyzing the defects in mice with a mutation in human gene counterparts is the most direct and cost-efficient approach for understanding human development, physiology, and diseases. Having been extensively used to extrapolate the observed physiological alternation from the mouse model to human disease, a mutant mouse library with individual mice carrying a mutation in one of all genes are highly demanded in both academic and biopharmaceutics.
Presently, approximate 66% of the protein coding genes in the mouse genome have been disrupted by random gene trap insertions (De-Zolt, et al., 2006). It is thought that only 60% of mouse genes can be effectively targeted by current gene traps technology. The limitation of gene trap technologies currently available is evident by the fact that after the percentage of genes trapped in the entire genome reaching the 60% limitation, the chance of trapping new genes in ES cell decreases exponentially. Thus, further trapping genes beyond the 60% limit will be non-effective and impractical since the chance of trapping new genes will drop down to zero long before completely trapping every single gene in the entire mouse genome (Skarnes, et al., 2004; 2005). The difficulty of trapping the remaining 40% of genes could be attributed to the inherited bias of retrovirus-mediated integration, developed by Lexicon Technology, that selectively targets genes actively expressed in ES cells (Scheridin, 1990). Alternatively, the structure of genes may also impede the accessibility of trapping cassette as revealed by transmembrance domain containing proteins (De-Zolt, et al., 2006). Regardless, a major limitation of generating a gene trap library covering the entire genome is posed by the adverse effect of exclusively relying on drug selections to obtain targeted clones in all gene trap practices conducted so far.
Fraser discloses a “piggyBac constructs in vertebrates”. The piggyBac transposon is disclosed herein as an extremely versatile helper-dependent vector for gene transfer and germ line transformation in a wide range of vertebrate species. PiggyBac mobility is demonstrated using an interplasmid transposition assay that consistently predicts the germ line transformation capabilities of this mobile element in several species. Both transfected COS-7 primate cells and injected zebrafish embryos supported the helper-dependent movement of tagged piggyBac element between plasmids in a cut-and-paste fashion.
Manfred (U.S. Pat. No. 5,922,601; July/1999) discloses a “High efficiency gene trap selection of regulated genetic loci”. A gene trap construct for identification of genes whose activity is regulated upon a cellular transition event which comprises in downstream sequence: (i) a cassette having a functional splice acceptor, a translation stop sequence and an internal ribosome entry site and (ii) a promoterless protein coding sequence encoding at least one polypeptide providing positive and negative selection traits. A method for identification of genes whose activity is regulated upon a cellular transition event by introducing the gene trap construct into a cell and observing expression of the positive and/or negative selection traits before and after the transition event.
Zambrowicz (U.S. Pat. Nos. 6,436,707 and 6,080,576) discloses a “Vectors for gene mutagenesis and gene discovery”. Novel vectors are described that incorporate, inter alia, a novel 3 gene trap cassette which can be used to efficiently trap and identify previously unknown cellular genes. Vectors incorporating the described 3 gene trap cassette find particular applications in gene discoveries and in the production of mutated cells and animals.
Ong discloses a “complementation trap”. The methods and DNA constructs are provided for detection and manipulation of a targeted eukaryotic gene whose expression is restricted to certain tissues or specialized cell types. The methods include transforming a cell with a first indicator component under the control of a promoter selected for its restricted expression in a particular cell or tissue. The cell is also transformed with a gene trap vector encoding a second indicator component. The cell is allowed to differentiate to produce specialized cell or tissue which is monitored for expression of both the first and second indicator components, thereby detecting a gene into which the trap vector has integrated and is expressed in the same cell or tissue type as the selected promoter.
All of above prior arts using plasmid vectors, transposons, or viral vectors that can be performed in vertebrates and mammals to achieve genetic manipulations, such as the piggyBac transposon-mediated gene disruption and transgenesis, are exclusively mediated either by a drug (antibiotics) selection, reporter gene selection or specific phenotype (Tyrosinase I & II mutations) selection to manipulate cells or animals.
Applying the drug-mediated selection circumvents the difficulty of obtaining targeted clones as the efficiency of gene targeting is usually very low. It results in selectively targeting to actively expressed genes or genes in active chromosomal regions. Consequently, the prior arts in genetic modification are ineffective in manipulating genes that are silent or suppressed at the time of chromosomal modification.
Maintaining ES cell in a pluripotent status requires repression of some key genes crucial in determining the fate of ES cells as they differentiate toward a defined cell lineage. It has been recently shown that the mechanism of restricting expression of such genes in ES cell for maintaining ES cell pluripotency is governed by the polycomb repressive complex (PRC) (Boyer, et al., 2006; Bernstein, et al., 2006; Lee, et al., 2006). Since the PRC forms a higher order of chromatin complex structure in the promoter region of certain genes to assure their silent status, a drug-dependent gene trap approach is unlikely to be succeeded in harvesting ES clones trapped in such gene loci. This is a major obstacle encountered by all of gene trap technologies currently available. As silenced genes may constitute a large portion of the “untargetable” genes (about 40% of the entire mouse genes), there is a critical need in developing a high efficient drug-independent gene trap system to surpass this difficulty.
To circumvent the aforementioned difficulties, there remains a need for new methods to reach the goal of unbiased gene targeting without drug selection, particularly the methods that perform highly efficient chromosomal insertion and are able to target the silent regions on chromosomes.